Many proposals have been made for methods of transferring a gene such as DNA and RNA into cells, but still cannot be said to be satisfactory, and are problematic in that the transfer techniques per se are complex and also low in transfer efficiency. For example, there has been proposed a method for transferring a gene into cells utilizing a virus (e.g., refer to Non-Patent Document 1). However, when a virus is used, a gene to be transferred must be purified with a complicated method, and there may be a risk that the virus may transfer a gene into cells or tissues that are not intended. Also, it is required to consider a risk of biohazard due to the virus.
In addition, there has been proposed a calcium phosphate method (e.g., refer to Non-Patent Document 2). This is a method in which calcium phosphate particles containing DNA are produced and taken up into cells due to endocytosis. However, this method is problematic in that the procedures are complicated and low in transfer efficiency. As an improved method thereof, there has been proposed a method in which a substance is taken up into cells utilizing a reagent such as a cationic liposome and a cationic polymer (e.g., refer to Non-Patent Documents 3 and 4). The cationic liposome is problematic in that it is low in gene transfer efficiency because it is easily decomposed by a lysosome and the like in a cell after being taken up into the cell through the endocytosis process. Further, synthesis of molecule species used in the liposome is complicated, and complicated production methods are required including ultrasonic treatment used for liposome formation. As a cationic polymer for transferring a gene, one having a straight chain structure of polyethyleneimine is said to be efficient. However, when polyethyleneimine is synthesized from commercially available raw materials, it is very difficult to obtain the straight chain molecule since ring-opening polymerization of ethyleneimine is required which yields a branched structure with primary, secondary and tertiary amines.
Besides, a method called as electroporation is frequently used (for example, refer to Patent Document 1). This is a method in which a high voltage in pulse form is applied to a suspension of a gene together with cells so that the gene contained in the suspension is allowed to be taken up into cells. This method has a wide range of application and is high in gene transfer efficiency, but cannot transfer the gene without cell death since the gene transfer efficiency has a proportional relation with cell lethality. That is, it is problematic in that when a pulse condition is improper, not only the target substance cannot be transferred into the cell, but also the cell may die. Also, electric conductivity of the cell suspension must be lowered in order to prevent so-called towing which is electric discharge into a solution.
In addition, when a gene is transferred into cells using a reagent or virus, it is difficult to transfer it into a specified site, and particularly it is very difficult to transfer it into just a targeted site of the cells that are attached to a petri dish on which the substance to be transferred easily diffuses due to the medium contained therein. Similarly, even with electroporation, it is difficult to transfer the gene into the attached cells without peeling of cells or cell fusion.
Therefore, the methods in which genes are transferred into cells using a conventional virus or reagent have technical problems such that the procedure itself is very complicated and difficult to practice. Also, particularly when a medium is present, there has been a problem such that a solution of a gene to be transferred is diffused but cannot be focused on a site where target cells are present, while peeling of cells or cell fusion easily occur.
[Conventional Technical Documents]
[Patent Documents]
    Patent Document 1: U.S. Pat. No. 4,945,050[Non-Patent Documents]    Non-Patent Document 1: D. Yu, T. Shioda, A. Kato, M. K. Hasan, Y. Sakai, Y. Nagai, Genes Cells, 1997 vol. 2(7), p. 457-66    Non-Patent Document 2: F. L. Graham, A. J. Van Der Eb, Virology, 1973, vol. 52, p 446-467.    Non-Patent Document 3: P. L. Felgner, T. R. Gadek, M. Holm, R. Roman, H. W. Chan, M. Wenz, J. P. Northrop, G. M. Ringold, M. Danielsen, Proc. Natl. Acad. USA, Vol. 84, p 7413-7417.    Non-Patent Document 4: D. Goula, J. Remy, P. Erbacher, M. Wasowicz, G. Levi, B. Abdallah, B. Demeneix, Gene. Therapy, 1998, vol. 5, p 712-717.